13334
Biochemistry 1995, 34, 13334- 13342
Phospholipid Binding and Lecithin-Cholesterol Acyltransferase Activation Properties of Apolipoprotein A-I Mutants? Paul Holvoet,*J Zhian Zhao,$ Berlinda Vanloo,$ Rita Vos," Els Deridder,* Ann Dhoest,$ JosCe Taveirne,! Els Brouwers,$ Eddy Demarsin,* Yves Engelborghs," Maryvonne Rosseneu,! DCsirC Collen,* and Robert Brasseurl Center for Molecular and Vascular Biology, University of Leuven, B-3000 Leuven, Belgium, Department of Clinical Chemistry, A. Z. &-Jan, Brugge, Belgium, Laboratory of Chemical and Biological Dynamics, University of Leuven, B-3000 Leuvenm, Belgium, and Centre de Biophysique MolCculaire Numtrique, Facultt de Sciences Agronomiques de Gembloux, Belgium Received June 9, 1995; Revised Manuscript Received August 21, 1995@
ABSTRACT: Recombinant human apolipoprotein A-I (apo A-I) and three deletion mutants: apo A-I(ALeu4Leu'26), apo A-I(AGlu'39-Leu'70), and apo A-I(AAla190-Gln243),purified from the periplasmic space of Escherichia coli, were studied. The rate of turbidity decrease following mixing of apo A-I(AAla190Gln243)with dimyristoylphosphatidylcholine(DMPC) vesicles at 23 "C was 10-fold lower than that of the other apo A-I proteins, confirming that the carboxy-terminal region of apo A-I plays a role in rapid lipid binding. The Stokes radii of reconstituted high-density lipoproteins (rHDL), containing dipalmitoylphosphatidylcholine and cholesterol, were larger for the three apo A-I mutants [6.3 nm for apo A - I ( A L ~ U ~ ~ - L ~ 6.1 U I ~nm ~ ) for , apo A - I ( A G ~ U ~ ~ ~ - Land ~ U 6.5 ' ~ ~nm ) , for apo A-I(AAla190-Gln243)] than for intact apo A-I (5.0 nm). The mutant rHDL all contained 4 apo A-I molecules per particle as compared to 2 for intact apo A-I. Circular dichroism measurements revealed 8 a-helices per apo A-I molecule, 5 per apo A - I ( A L ~ u ~ ~ - L ~ u6 'per ~ ~ apo ) , A - I ( A G ~ u ~ ~ ~ - L ~ uand I ~ O4 )per , apo A-I(AAlaI9OGln243)molecule as compared to predicted values of 8, 5, 6, and 6 a-helices, respectively. The catalytic efficiencies for activation of lecithin-cholesterol acyltransferase (LCAT) were 2-fold lower for apo A - I ( A L ~ u ~ ~ - L ~ 11 u ' -fold ~ ~ ) ,lower for apo A - I ( A G ~ U I ~ ~ - L and ~ U 8-fold ~ ~ ~ ) lower , for apo A-I(AAla190than for intact apo A-I, as a result of a significant decrease of V,,, but not of K, values. In aggregate, these data suggest that deletion of the amino-terminal or of the central domains of apo A-I has little effect on lipid binding, whereas deletion of the carboxy-terminal domain reduces the rate but not the extent of lipid binding. Furthermore, deletion of the central domain [in apo A - I ( A G ~ U ~ ~ ~ - L or ~U'~~)] conformational modifications in the central domain [in apo A-I(AAla190-G1~243)] result in a decrease of the rate of LCAT activity that is independent of the binding of LCAT to rHDL.
Low plasma levels of high-density lipoproteins (HDL)' and of their major protein component, apolipoprotein A-I (apo A-I), correlate with an increased risk for coronary heart disease (Gordon & Rifkind, 1989), and family and twin studies have suggested that decreased HDL levels are partially heriditary (Christian et al., 1990; De Backer et al.,
1986; Hunt et al., 1989; Pometta et al., 1979). In addition, HDL and their apolipoproteins increase the net efflux of cellular unesterified cholesterol (Daniels et al., 1981;Fielding & Fielding, 1981; Oram et al., 1981), and remove excess cholesterol from peripheral (nonhepatic) cells (Glomset, 1968), which may explain the inverse correlation between risk of coronary heart disease and HDL levels (Miller et al., 1985). 'This study was supported by the Nationaal Fonds voor Wetenschappelijk Onderzoek (Project 3.0063.94). Cholesterol esterification, a requirement for its transport * Correspondence should be addressed to this author at the Center from peripheral cell membranes to the liver, is catalyzed by for Molecular and Vascular Biology, University of Leuven, Campus LCAT, a reaction that is greatly stimulated by apo A-I Gasthuisberg, 0 & N Herestraat 49, B-3000 Leuven, Belgium. Telephone: 32-16-34.57.72. Fax: 32-16-34.59.90. (Fielding et al., 1972). LCAT plays a role not only in the Center for Molecular and Vascular Biology, University of Leuven. reesterification of HDL cholesterol but also in the regulation A. Z. St.-Jan. of the HDL level, as suggested by the greatly decreased HDL l 1 Laboratory of Clinical and Biological Dynamics, University of levels in familial LCAT deficiency (Funke et al., 1991a) and Leuven. FacultC de Sciences Agronomiques de Gembloux. in fish-eye disease (Funke et al., 1991b). Abstract published in Advance ACS Abstracts, October 1, 1995. Apo A-I is synthesized as a prepropeptide; it is cotransI Abbreviations: apo A-I, apolipoprotein A-I; apo A - I ( A L ~ u ~ ~ LeuIz6),apo A-I mutant with the L e ~ ~ ~ - L segment e u ' ~ ~ deleted; apo lationally cleaved to pro-apo A-I, and during secretion A - I ( A G ~ U ~ ~ ~ - Lapo ~U A-I ' ~mutant ~ ) , with the G l ~ ' ~ ~ - L segment eu'~~ cleaved to form the mature 243 amino acid apo A-I protein deleted; apo A-I(AAla'90-Gln243),apo A-I mutant with the Ala'90(Brewer et al., 1978). Folding of apo A-I into amphipathic Gln243segment deleted: DMPC, dimyristoylphosphatidylcholine; D P K , helices composed of hydrophilic and hydrophobic surfaces dipalmitoylphosphatidylcholine;EDTA, ethylenediaminetetraacetic acid: HDL, high-density lipoproteins; rHDL, reconstituted high-density (Segrest et al., 1974, 1990, 1992) has been demonstrated lipoproteins; LCAT, lecithin-cholesterol acyltransferase; CD, circular using complexes of phospholipids with plasma apo A-I and dichroism; FPLC, fast performance liquid chromatography; HPLC, with model peptides (Anantharamaiah et al., 1985; Fukuhigh-performance liquid chromatography: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. shima et al., 1980; Kroon et al., 1978; Nakagawa et al., 1985; @
0006-2960/95/0434- 13334$09.00/0
0 1995 American Chemical Society
Biochemistry, Vol. 34, No. 41, 1995 13335
Structure of Apo A-I in Mutant Reconstituted HDL
...
...........
&
epo A-I (AAlal~-GIn243)
I
11 ............... '!?...........
111 _.
........................... U
la1
.
IU
FIGURE 1: Schematic representation of the secondary structure of apo A-I proteins in rHDL. Open circles represent proline residues in /3-strands and closed circles tryptophan residues in the amino-terminal domain of apo A-I that are contributing to the fluorescence properties. For apo A-I(AAla190-Gln243), three possible conformations are illustrated: (I) with 6 helices bound to the edge of the disc; (11) with 4 helices bound to the edge of the disc and a hinged domain located between residues Asnlo2and LysI4O; or (111) with 4 helices bound to the and His162. edge of the disc and a hinged domain located between residues
Sparrow & Gotto, 1980; Srinivas et al., 1991; Vanloo et al., 1991). Based upon energy minimization calculations and infrared spectroscopy measurements, a general model was proposed for the association of apo A-I and phospholipids in discoidal complexes (Brasseur, 1991; Brasseur et al., 1990; Wald et al., 1990). According to this model, apo A-I contains 8 putative amphipathic a-helices that are oriented parallel to the acyl chains of the phospholipids, around the edge of the discs, with their hydrophobic surface toward the lipid core and with their hydrophilic surface toward the aqueous phase (Figure 1). The first amino-terminal domain (residues 44-63) has the lowest helical structure probability, while the second helix (residues 69-85) is not involved into a pair (Brasseur, 1991; Brasseur et al., 1990). The 6 carboxyterminal helical structures most likely form pairs of antiparallel a-helices stabilized by protein-protein interactions. A minimum length of 17-20 amino acids (5-6 helical turns) appears to be required for effective phospholipid binding and LCAT activation (Brasseur, 1991; Brasseur et al., 1990). However, the domains of apo A-I involved in phospholipid binding and/or LCAT activation remain unidentified. Two naturally occurring mutants, one with a deletion of Lyslo7 (Rall et al., 1984) and one with a substitution of Pro143with Arg (Utermann et al., 1984), have a 50% reduced ability to activate LCAT. Minnich et al. (1992) found that although binding of apo A - I ( A G ~ u ~ ~ ~ - Aapo ~ ~A-I(AMet148'~~), Glyls6), and apo A - I ( A A S ~ ~ ~ ~to -liposomes G ~ ~ ~ ~was ~ )5080% of normal, the two latter apo A-I mutants surprisingly did not form discoidal complexes. LCAT activation with these mutants was 47%, O S % , and 13% of normal. For apo A-I(AAS~~'~-G this~ was ~ ~ ~found ~ ) , to be due to a 15-fold reduction of the K , value, suggesting decreased binding of LCAT to the multilamellar vesicles obtained with this mutant. Sorci-Thomas et al. (1993) found that the binding of apo A - I ( A P ~ O ~ ~ - G ~ ~apo ' ~ OA) ,- I ( A p r ~ l ~ ~ - A l a ' ~and ~ ) , apo A-I(AP~O*~~to unilamellar vesicles was normal, but their LCAT activity was reduced 10-fold. However, from these data it was not clear whether the reduced LCAT
activation was due to reduced V,,, or to increased K,. Furthermore, because the composition of the complexes and the conformation of the apo A-I proteins in the respective complexes were not studied, it could not be excluded that reduced LCAT activation was due to conformational differences instead of deletion of a domain that is critical for LCAT interaction. However, Jonas et al. (1989) have previously demonstrated that conformational changes of intact apo A-I in larger discoidal rHDL, with decreased a-helical contents, already are associated with a 20-fold decrease of LCAT activation. Recently, Schmidt et al. demonstrated that deletion of the carboxy-terminal domain of apo A-I resulted in decreased in vivo lipoprotein association and thus in an increased clearance rate (Schmidt et al., 1995). In the present study, three mutants of apo A-I were selected in which putative phospholipid binding and/or LCAT activation domains were deleted. The three first amino-terminal a-helical repeats, overlapping with the putative LCATactivating domain proposed by Anantharamaiah et al. (1990), were deleted in apo A - I ( A L ~ u ~ ~ - L ~(Figure u ' ~ ~ )1). The central ( G l ~ ~ ~ ~ - Ha-helix, i s ' ~ ~ )with its adjacent P-strands, overlapping the G l ~ ' ~ ~ - A lputative a ' ~ ~ LCAT activation domain (Sparrow & Gotto, 1980), was deleted in apo A - I ( A G ~ U ' ~ ~ - L(Figure ~ U ~ ~1). ~ ) Finally, the two carboxyterminal a-helical repeats, overlapping the putative phosfragment that by itself could pholipid binding not activate LCAT (Sparrow & Gotto, 1980; Soutar et al., 1975), were deleted in apo A-I(AAla'90-Gln243) (Figure 1). In this study, we demonstrate that deletion of the AlalgOGln243carboxy-terminal domain from apo A-I reduces its rate of phospholipid binding, but does not prevent discoidal complex formation. Furthermore, deletion of the G l ~ l ~ ~ Leu170central domain or the Ala190-Gln243carboxy -terminal domain but not of the Leu44-Leu126amino-terminal domain results in a disruption of conformation of the apo A-I central domain and in a decrease of the rate of LCAT activation that is independent of LCAT binding.
13336 Biochemistry, Vol. 34, No. 41, 1995
EXPERIMENTAL PROCEDURES Oligonucleotide-Directed Mutagenesis and DNA Sequencing. All DNA manipulations were carried out essentially as described by Maniatis et al. (1982). Oligonucleotidedirected mutagenesis was performed by the gapped-duplex method of Kramer et al. (1984) using the p M d c vector system of Stanssens et al. (1989). This system employs phasmid (Le., phage/plasmid hybrid) vectors allowing cloning and site-directed mutagenesis as well as sequencing to be carried out in the same vector without recloning. Oligonucleotides were obtained by custom synthesis (Pharmacia, Brussels, Belgium). DNA sequences were determined using a primer walking strategy on a Pharmacia ALF DNA sequencer (Pharmacia, Uppsala, Sweden). Template DNA was purified by alkaline hydrolysis followed by a poly(ethylene glycol) precipitation step. The sequencing reactions were carried out using T7 DNA polymerase (Pharmacia) and a fast denaturation protocol as described elsewhere (Zhou et al., 1990). The reaction products were sized on 6% Hydrolink Long Ranger gels (AT Biochem, Malvern, PA) containing 1 x TBE buffer and run with 0 . 6 ~TBE buffer. The sequence was assembled using the IntelliGenetics Suite 5.4 program (IntelliGenetics, Palo Alto, CA). Construction of cDNA for Expression of Wild-Type Apo A-I and Apo A - I Mutants. The pMc-5-apo A-1 transfection vector, encoding wild-type recombinant apo A-I, for expression in E. coli under control of the tac-promoter and the PhoA signal peptide sequence was constructed starting from the plasmid pAI-3 (Cheung & Chan, 1983), the pUC-19 vector (Yanisch-Perron et al., 1985), and the pMa/c mutagenesis vector (Stanssens et al., 1989). The plasmid pAl3, obtained by insertion of the apo A-1 cDNA in the PstI site of pBR322 (Cheung & Chan, 1983), was a kind gift of Dr. L. Chan, Baylor College of Medicine (Houston, TX). The 961 bp PstI fragment from pAI-3, comprising the entire coding region and the signal peptide sequence of apo A-I, was ligated in the PstI-treated pUC-19 vector, resulting in the pUC-apo A-I vector. The 1010 bp KpnI-Hind111 fragment from pUC-apo A - I was ligated in the KpnI- and HindIII-treated pMc-5 vector, resulting in the pMc-5-int vector, containing both the PhoA signal peptide and the apo A-I signal peptide coding sequences. A 43 bp fragment,
dGATGAACCACCACAGAGTCCATGGGATCGAGTGAAGGATCTGG, was inserted in the KpnI (blunt-ended)MscI-treated pMc-5-int, resulting in the pMc-5-apo A - I vector, in which the apo A-I signal peptide coding sequence was deleted. In addition, in order to increase the efficiency of mutagenesis, 5 silent mutations were simultaneously introduced in the 43 bp apo A-I coding sequence: the CCC codons for Pro3, Pro4, and Pro7 were replaced with CCA; the AGC codon for Ser6 was replaced with AGT; and the GAC codon for Asp13was replaced with GAT. The pMc5-apo A - I cDNA thus expressed wild-type apo A-I under the control of the tac-promoter in E. coli (Figure 2). Deletion mutagenesis with the 47-mer deoxynucleotide dAGCTTCTGGCGCGCGCCCTClTGGTTC A G C T G m CCCAAGGCGGA was performed on the pMc-5-apo A-I vector to delete the coding sequence, resulting in the pMC-5-apo A-I(ALeu44-Leu126)vector for expression of apo A - I ( A L ~ L I ~ ~ - L ~inUE. ~ *coli ~ ) (Table 1). Deletion mutagenesis with the 36-mer oligodeoxynucleotide dGGCCAAGCGCTGGCGTTGCAGCT CGTGAAGCTTCTG was performed on the pMc-5-up0 A-1 vector to
Holvoet et al. delete the G l ~ ~ ~ ~ - L sequence, e u " ~ resulting in the pMc-5Apo A - I ( A G ~ U ~ ~ ~ - Lvector ~ U ~ ' ~for ) expression of apo A-I(AG~U'~~-L in ~E.Ucoli. ~ ~ ~By ) substitution of the CTG codon for Leu134with the CTT codon for Leu, an additional Hind111 restriction site was created (Table 1). Deletion mutagenesis wih the 48-mer oligodeoxynucleotide dGGAAGGTGGGCGACGGCGAGCGCCTCACAGT CTGGCACCACCGTTCTC was performed on the pMc-5apo A-I vector to delete the Ala19n-Gln243coding sequence, resulting in the pMc-5-apo A - I (AAla19n-Gln243)vector for expression of apo A-I(AAla19n-Gln243)in E. coli (Table 1). All cDNA constructs were confirmed by DNA sequencing. Expression in E. coli and Cell Fractionation. E. coli WK6 host cells were grown in 2YT medium to an optical density at 600 nm of approximately 0.4, at which point isopropylP-D-thiogalactopyranoside was added to a final concentration of 0.4 mM. Cells were further incubated at 37 "C for 2 h, during which the optical cell density increased to 10l2cells/ L. The cell suspensions (10 L) were centrifuged at lOOOOg for 10 min, the supernatant was removed, and periplasmic cell fractions were prepared as described previously (Koshland & Botstein, 1980). The pellet is treated with 70 mL of 0.02 M Tris-HC1, pH 8.2, containing 27% sucrose. Thereafter, 800 p L of 0.5 M EDTA and 4 mL of a 2 mg/mL lysozyme solution are added. The mixture is incubated for 30 min on ice. Thereafter 1.2 mL of 1 M CaC12 is added. Finally, the pellet is centrifuged for 15 min, the supematant is harvested, and (NH4)2S04 is added to a final concentration of 1 M. Purification. All proteins were purified to homogeneity by a combination of hydrophobic interaction chromatography, ion-exchange chromatography on Q-Sepharose, and gel filtration. The supematant is applied to a 2.5 x 12 cm phenyl-Sepharose column. Bound apo A-I proteins were eluted by applying a 1-0 M (NH&S04 gradient (total volume 190 mL). Apo A-I proteins, assessed in ELISA, eluted at 0.1 -0.15 M salt. The apo A-I-containing fractions were pooled, dialyzed against 0.02 M Tris-HC1, pH 8.2, containing 7 M urea, and applied to a 2.5 x 6 cm Q-Sepharose column. Bound apo A-I proteins were eluted by applying a 0-0.1 M NaCl gradient (total volume 100 mL). Apo A-I proteins eluted at 0.04-0.06 M salt. The apo A-I-containing fractions were concentrated with PEG 20 000and applied to a 1.6 x 60 cm Superdex 75 Prep Grade column, equilibrated with 0.02 M Tris.HC1, pH 8.2, containing 0.15 M NaC1. Circular Dichroism Spectrometry. Circular dichroism spectra of lipid-free apo A-I proteins in solution and of apo A-I proteins in discoidal apo A-I/phospholipid/cholesterol complexes were measured at increasing temperatures with a Jasco 5600 spectropolarimeter (Japan Spectroscopy, Tokyo, Japan) at wavelengths between 200 and 250 nm, using 6 p M sample solutions and a 1 mm path length cuvette. Temperatures were increased from 25 to 85 "C at a rate of 3 "C/5 min. Backgrounds were measured at 250 nm for 5 min, followed by measurements of the a-helical content at 222 nm for 5 min. For all apo A-I proteins, the a-helical content could be restored upon cooling of the solutions overnight. The fraction of a-helices in the secondary structure of the apo A-I proteins was estimated from the molar ellipticities at 222 nm ([el222 = (-30 300 x fH) 2340) where fH is the fraction of a-helical structure (Chen et al., 1972). DMPC-Apo A - I Assembly. Kinetic turbidimetric measurements were carried out as described by Pownall et al.
Biochemistry, Vol. 34, No. 41, 1995 13337
Structure of Apo A-I in Mutant Reconstituted HDL P
P
H pUC-ApoA-l
Kpnl-Hindlll digestion
Kpnl-Hlndlll dlgortion
K
El.
Yp .M
pr= \y ...
dGATGM TGG
I FIGURE 2: Construction of the pMc-5-upo A-I transfection vector. The 961 bp PstI fragment from pA1-3 was ligated in the PstI-treated pUC-I9 vector, resulting in the pUC-up0 A-I vector. The 1010 bp KpnI-Hind111 fragment from pUC-up0 A-I was ligated in the KpnI- and HindIII-treated pMc-5, resulting in the pMc-5-int vector, containing both the PhoA signal peptide and the upo A-I signal peptide coding sequences. A 43 bp fragment, dGATGAACCACCACAGAGTCCATGGGATCGAGTGAAGGATCTGG,was inserted in the KpnI (bluntended)-MscI-treated pMc-5-int vector, resulting in the pMc-5-Apo A-I vector. In this cloning step, the upo A-I signal peptide coding sequence was removed. In addition, 5 silent mutations were simultaneously introduced, as described under Experimental Procedures. Hatched boxes represent the mature upo A-I cDNA sequence, open boxes the upo A-I signal peptide, black boxes the PhoA signal peptide, and open triangles the fuc promoter. Restriction endonucleases indicated are as follows: EI, EcoRI; H, HindIII; K, KpnI; M, MscI; P, PstI; X, XbuI.
(198 1). DMPC and apo A-I proteins were dissolved at molar concentrations of 600 and 6 p M in 0.01 M Tris-HC1 buffer, pH 7.4, containing 8.5% KBr, 0.01% sodium azide, and 0.01% EDTA and incubated at 23 "C for 10 min before mixing of equal volumes of the reagents. The lipid-protein association was followed by monitoring the rate of disappearance of liposomal turbidity at 325 nm. Pownall et al. (1981) defined a rate constant k1/2= l/t1/2, where t112 is the time required for the decrease of half of the relative turbidity [(A0 - A-JAol. Preparation of Discoidal Apo A-I/DPPC/Cholesterol Complexes. Complexes of the apo A-I proteins with dipalmitoylphosphatidylcholine(DPPC, Sigma) and cholesterol, at an apo A-I/DPPC/cholesterol ratio of 1/3/0.15 (w/ w/w), were prepared using the cholate dialysis procedure (Matz & Jonas, 1982; Vanloo et al., 1991). The mixture was incubated at 43 "C for 16 h, and cholate was removed by extensive dialysis. Complexes were isolated by gel
filtration on a Superose 6 HR column equilibrated with 20 mM Tris-HC1 buffer, pH 8.1, containing 0.15 M NaCl and 0.02 mg/mL sodium azide, in a FPLC system (Waters). One milliliter fractions were collected. The size of these complexes was estimated by comparison of their migration position on native 4- 15% gradient polyacrylamide gels with that of standard proteins: thyroglobulin (Stokes radius of 8.5 nm), apoferritin (6.1 nm), catalase (5.2 nm), and lactate dehydrogenase (4.1 nm) (Pharmacia). The number of apo A-I molecules per lipoprotein particle was determined following cross-linking of apo A-I with bis(su1fosuccinimidyl) suberate (final concentration 1 mM) for 1 h, as previously described (Swaney, 1986), and separation of cross-linked proteins by SDS-PAGE on 4- 15% gradient gels. The extent of oligomer formation was estimated by comparison with cross-linked plasma apo A-I (Swaney, 1986). The chemical composition of the isolated complexes was assayed as follows: the levels of phospholipid and
13338 Biochemistry, Vol. 34, No. 41, 1995
Holvoet et al.
cholesterol were determined using commercial enzymatic kits (BiomCrieux, France, for phospholipid; Boehringer Mannheim, Germany, for cholesterol), and the concentrations of apo A-I were determined according to Bradford (1976). Fluorescence Analysis. Corrected steady-state fluorescence spectra and intensities were obtained on a SPEX spectrofluorometer (Fluorolog 1691). Excitation was carried out at 295 nm where the contribution of tyrosyl fluorescence to the total intensity is minimal. Fluorescence was measured with a 2 mm slit width in quartz cuvettes with optical path lengths equal to 1 and 0.4 cm and wavelength resolutions of 7.2 and 3.6 nm for the emission and the excitation wavelengths, respectively. Excitation was vertically polarized and fluoresence detected with a polarizer at a 54" angle to reduce the influence of the fluorescence depolarizatoin and Brownian motion on the detected intensity. The spectra were corrected for the wavelength dependence of the emission monochromator and the photomultiplier and for background intensities as described previously (Roberts, 198 1). In the fluorescence quenching experiments, the spectra were recorded at increasing concentrations of KI. The results were analyzed by the Stern-Volmer equation as modified by Lehrer (Lehrer, 1971): Fd(F0 - F) = l/fa (l/fKsv)(l/[KI]), where FO and F are the fluorescence intensities oberved in the absence or in the presence of a given concentration of KI, respectively, and fa is the fraction of quenchable fluorescence (Jonas et al., 1993). The SternVolmer constant, KSV,is an index of the accessibility of tryptophan residues to the aqueous phase. Pur$cation of LCAT. LCAT was purified 5 100-fold from plasma and ultracentrifuged at densities of 1.21 and 1.25 g/mL, by chromatography on Affi-Gel Blue and DEAESephadex (Matz & Jonas, 1982). Remaining impurities (65% of total protein), detected by SDS -PAGE electrophoresis and immunoblotting, essentially consisted of albumin. The enzyme was dialyzed against 10 mM Tris-HC1 buffer, pH 7.6, containing 5 mM EDTA, and solutions were stored at -80 "C. Kinetics of LCATActivation by Discoidal Apo A-I/DPPC/ Cholesterol Complexes. LCAT activation by discoidal apo A-I/DPPC/cholesterol complexes was monitored by incubation of the equivalent of 10-40 p M unesterified cholesterol, with a final concentration of 50 nM LCAT (Vercaemst et al., 1989). The reaction was carried out in 10 mM Tris.HC1 buffer, pH 8.0, containing 5 mM EDTA, 0.15 M NaC1,4-16 mg/mL delipidated bovine serum albumin and 6 mM P-mercaptoethanol. After preincubation for 20 min at 37 "C, LCAT was added. The mixture was incubated at 37 "C for 10-180 min, whereafter the reaction was arrested by extraction of cholesteryl esters with hexane/2-propanol (3/ 2, v/v) containing cholesteryl heptadecanoate as an internal standard. Under these reaction conditions, less than 20% of the cholesterol was esterified. Generated cholesteryl esters were quantified by isocratic HPLC on a reversed-phase ZORBAX ODS column, eluted with acetonitrile/2-propanol (50/50,v/v), as described previously (Vercaemst et al., 1989). Initial activation rates were obtained from plots of the concentration of generated cholesteryl esters versus time, and the kinetic parameters, K, (expressed in micromolar cholesterol) and V,,, (expressed in nanomoles of cholesteryl esters generated per hour), were determined by linear regression analysis from Lineweaver-Burk plots.
+
Table 1: Amino Acid Compositions and Molecular Masses of Intact ADOA-I and ADOA-I Mutants
no. of compound amino acids plasma apo A-I 243 recombinant apo A-I 243 apo A-I(ALeu44-Leu126) 160 apo A - I ( A G I U ' ~ ~ - L ~ U ~ ~21 ~ )1 apo A-I(AAla'90-Gln243) 189
mol massu (kDa) 28 28 18 24.4 22
mol massb (kW 28 28 18.4 24.1 21.7
a Molecular mass calculated on the basis of the amino acid composition. Molecular mass calculated from the migration distance on 10- 15% SDS-polyacrylamide gels.
-
--
94 67 43
30 20
14.4
--
-
Ly-
-
v-
FIGURE 3: SDS-PAGE of purified apo A-I proteins on 10-15% gradient gels. Proteins (5 pg per lane) were stained with Coomassie Brilliant Blue. Lane 1, plasma apo A-I; lane 2, recombinant apo A-I; lane 3, apo A - I ( A L ~ u ~ ~ - L ~ ulane I ~ ~4,) ;apo A-I(AGIu'~~Leul'O); lane 5 , apo A-I(AAla'90-Gln243); and lane 6, protein calibration mixture consisting of phosphorylase b ( M , 94 kDa), albumin ( M , 67 kDa), ovalbumin ( M , 43 kDa), carbonic anhydrase ( M , 30 kDa), trypsin inhibitor ( M , 20 kDa), and a-lactalbumin ( M , 14.4 kDa).
RESULTS The predicted secondary structure of apo A-I (Brasseur et al., 1990; Brasseur, 1991) is illustrated in Figure 1. The predicted number of a-helices per molecule are 8 for intact apo A-I, 5 for apo A - I ( A L ~ u ~ ~ - L ~ u6' *for ~ ) apo , A-I(AG l ~ ' ~ ~ - L e u and ' ~ ~ 6) ,for apo A-I(AAla'90-Gln243). Wild-type apo A-I and the apo A-I mutants (Table 1) were expressed in the periplasmic space of E. coli cells, and purified to homogeneity as evidenced by their migration as single bands on 10- 15% SDS-polyacrylamide gels (Figure 3). Wild-type apo A-I migrated at exactly the same position as plasma apo A-I. Molecular masses of the apo A-I mutants, calculated from a plot of the logarithm of the molecular mass of the standard proteins vs the migration distance, were 18.4 kDa for apo A - I ( A L ~ u ~ ~ - L ~ u24.1 '*~), kDa for apo A - I ( A G ~ U ' ~ ~ - L ~and U ~21.7 ~ ~ )kDa , for apo A-I(AAla190-Gln243)(Table 1), and thus were in agreement with those calculated on the basis of the respective amino acid compositions. The rates of turbidity decrease following mixing of apo A-I moieties with DMPC vesicles (Table 2) were 10-fold lower for apo A-I(AAla190-Gln243)than for plasma apo A-I, recombinant apo A-I, apo A - I ( A L ~ u ~ ~ - L ~ uor' ~ apo ~), A - I ( A G ~ U ' ~ ~ - L ~suggesting U ~ ~ ~ ) , that the Ala190-Gln243 domain is determining the rate of lipid binding. However, with apo A-I(AAla190-Gln243)as with the other apo A-I proteins, more than 85% of the turbidity was cleared, thus suggesting that the extent of phospholipid binding was not affected by the deletion of the carboxy-terminal domain.
Biochemistry, Vol. 34, No. 41, 1995 13339
Structure of Apo A-I in Mutant Reconstituted HDL Table 2: Rate Constants for Decrease of Turbidity following Mixing of Apo A-I Proteins with DMPC" compound plasma apo A-I recombinant apo A-I apo A-I(ALeu44-Leu'26) apo A - I ( A G ~ U ~ ~ ~ - L ~ U ' ~ ~ ) apo A-I(AAla'y0-Gln243) a
k1,2 (min-I)
0.20 40.01 0.19 40.01 0.22 f 0.03 0.17 f 0.02 0.02 & 0.002
The data represent means & SD of 3 independent determinations.
The a-helical contents for the different apo A-I proteins in aqueous solution, as determined by CD scanning, were 48% for apo A-I, 40% for apo A-I(ALeu44-Leu126), 45% for apo A - I ( A G ~ u ~ ~ ~ - L ~and u ~48% ~ O )for , apo A-I(AAlaI9OGln243). SDS-PAGE of apo A-I proteins in aqueous solution, cross-linked at 6 pM final concentrations with bis(su1fosuccinimidyl) suberate, revealed '25% oligomers for all apo A-I proteins (not shown). These data suggested that the decrease of the rate of phospholipid binding of apo A-I(AAla190-Gln243)was not due to increased self-association. Discoidal apo A-I/DPPC/cholesterol complexes were obtained with all apo A-I proteins. Recoveries of apo A-I proteins in discoidal complexes after gel filtration were 90% for wild-type apo A-I (fractions 22-30 with maximum at fraction 27), 80% for apo A-I(ALeu44-Leu126)(fractions 1828 with maximum at fraction 23), and 70% for apo A - I ( A G ~ u ~ ~ ~ - L (fractions ~ u ~ ~ O ) 18-30 with maximum at fraction 25) and for apo A-I(AAla190-Gln243)(fractions 1828 with maximum at fraction 21). Top fractions (4 mL) were further analyzed. The apo A-Vphospholipidkholesterol molar ratios, calculated from the measured protein, phospholipid, and cholesterol levels, were 1/110/10 for intact apo A-I (identical for plasma apo A-I and recombinant apo A-I), 1/80/10 for apo A-I(ALeu44-Leu126), and 1/80/12 for both apo A - I ( A G ~ U ~ ~ ~ and - L apo ~ U A-I(AAla190-Gln243). ~~~) The complex particles obtained with the three apo A-I mutants had similar sizes but were larger than those obtained with intact apo A-I, whereas the sizes of the complexes obtained with plasma apo A-I and recombinant apo A-I were identical (Figure 4, Table 3). Cross-linking of the apo A-I molecules within the discoidal apo A-I/DPPC/C particles revealed 4 apo A-I molecules per particle for all apo A-I mutant rHDL as compared to 2 for intact apo A-I (Figure 5, Table 3). The phospholipid surface was calculated from the circumference of the disc using the measured diameter minus 3 nm, i.e., 2 x radius of an a-helix, multiplied with a height of the disc of 3.8 nm. The number of phospholipid molecules was calculated from the calculated surface divided by 0.45 nm2, the surface area per condensed phospholipid molecule. The calculated apo A-Yphospholipid molar ratios were 1/108 for intact apo A-I, 1/75 for apo A-I(ALeu44-Leu126), 1/71 for apo A - I ( A G ~ u ~ ~ ~ - L ~and u ~ ~1/79 O ) ,for apo A-I(AAla190Gln243),respectively, and were thus in agreement with the ratios calculated on the basis of the composition of the rHDL. The a-helical contents of the apo A-I proteins in discoidal apo A-VDPPC/cholesterol complexes were 74% for apo A-I, 75% for apo A-I(ALeu44-Leu126),60% for apo A-I(AG~uI~~Leu'70), and 52% for apo A-I(AAla190-Gln243). Thus, numbers of a-helices per apo A-I molecule, calculated from the a-helical content determined by CD scanning and from the protein length assuming a length of 22 amino acids per a-helical repeat (Brasseur et al., 1990; Segrest et al., 1974), were 8 for intact apo A-I, 5 for apo A-I(ALeu4-LeuIz6), 6
8.5
6.1
5.2
4.1
Stokes Radius (nm) FIGURE4: Determination of Stokes radii of rHDL by native polyacrylamide gel scanning. rHDL particles, isolated by gel filtration on a Superose 6HR column, were subjected to electrophoresis on 4- 15% gradient polyacrylamide gels under nondenaturing conditions. The protein calibration mixture contained thyroglobulin (Stokes radius of 8.5 nm), apofenritin (6.1 nm), catalase (5.2 nm), and lactate dehydrogenase (4.1 nm). Because the sizes of plasma apo A-I and recombinant apo A-I rHDL were identical, only the gel scan for recombinant apo A-I rHDL is presented.
for apo A - I ( A G ~ U ~ ~ ~ - Land ~ U 4~ 'for ~ ) apo , A-I(AAlaI9OGln243)(Table 3). These values are in agreement with the predicted values (Brasseur et al., 1990) for intact apo A-I, apo A-I(ALeu44-Leu126),and apo A - I ( A G ~ U ~ ~ ~ - but L~U~~~), are lower for apo A-I(AAla190-Gln243)(Table 3). The T , values, Le., temperature values at which the a-helical content is reduced by 50%, were 68,68,65, and 62 OC, respectively, further indicating that stable complexes were obtained with all apo A-I proteins. The fractions of the phospholipid surfaces that are covered by a-helix were calculated as the total phospholipid surface divided by 4 nm2, the surface area of an a-helix that contains 16 amino acids. The calculated fractions were 0.77 for intact apo A-I, 0.70 for apo A-I(ALeu44-Leu126),and 0.87 for apo A - I ( A G ~ U ~ ~ ~ - L ~ U ' ~ ~ ) , but only 0.53 for apo A-I(AAla190-Gln243)(Table 3). Binding of all apo A-I proteins to DPPC was associated with a shift of the Trp fluorescence maxima to lower wavelengths from 337 to 332 nm for intact apo A-I (both for plasma apo A-I and for recombinant apo A-I), from 338 to 329 nm for apo A-I(ALeu44-Leu126), from 339 to 333 nm for apo A - I ( A G ~ U ' ~ ~ - L and ~ U ~from ~ ~ )335 , to 332 nm for apo A-I(AAla190-Gln243)(Table 4), suggesting that lipid binding is associated with translocation of Trp residues to a more apolar environment. The quenching parameters KSV andf, are summarized in Table 4. For totally exposed Trp residues, in the absence of electrostatic or viscosity effects, Ksv = 12 M-I andf, = 1, whereas for totally protected Trp
13340 Biochemistry, Vol. 34, No. 41, 1995
Holvoet et al.
Table 3: Prouerties of Discoidal Apo A-IDPPCICholesterol Complexes ~~
properties Stokes radius (rimy apo A-I molecules per disc (n)b a-helical content (%o)c a-helices per apo A-I molecule (n)d estimated no. of a-helices per apo A-I (n)p fraction of phospholipid surface covered with a-helicesf
apo A-Ig 5.0 2
74 8 8 0.77
apo A-I(ALeu"''-Leu'26) 6.3 4 75 5 5 0.70
compound apo A-I(AG~u'"L~u''~) 6.1 4
60 6 6 0.87
apo A-I(AAla'90-Gln243) 6.5 4 52 4 6 0.53
a Size of rHDL particles determined by PAGE. The number of apo A-I molecules per rHDL particle was determined by SDS-PAGE after cross-linking with bis(sulfosuccinimidy1) suberate. The a-helical content was determined by CD scanning. The numbers of a-helices per apo A-I molecule were calculated from c and from the protein length assuming a length of 16 amino acids for the amphipathic helices and 6 amino acids for the adjacent /?-strands (Brasseur et al., 1990; Segrest et al., 1974). e Estimated number according to the model of Brasseur et al. (1990) (Figure 1). f The phospholipid surface was calculated from the circumference of the disc, using the measured diameter minus 3 nm, multiplied by the height of the disc of 3.8 nm. The area covered by an a-helix containing 16 amino acids was estimated to be 4 nm2. R Values for plasma apo A-I and recombinant aDo A-I were identical.
its fluorescence properties cannot be directly compared with those of the other apo A-I proteins. Thef, values for the single Trp8 residue in apo A - I ( A L ~ U ~ ~ - L ~were U I ~not ~) different from the fa values calculated for the other apo A-I proteins that all contain 4 Trp residues per molecule, thus indicating that the quenchable fluorescence represented on average 60% of the total fluorescence (Table 4) for all apo A-I proteins in rHDL. LCAT activation by all discoidal apo A-I/DPPC/cholesterol complexes obeyed Michaelis-Menten kinetics, as shown by linear Lineweaver-Burk plots of the inverse of the initial activation rate (llvo) versus the inverse of the cholesterol concentrations (l/[C]). The apparent kinetic parameters V,,, and K, and the V,,,/Km ratios for the different apo A-I/DPPC/cholesterol complexes are summarized in Table 5. Deletion of the G l ~ ' ~ ~ - L e uand "~ Ala'90-Gln243 domains resulted in a 1 1-fold and IO-fold decrease, respectively, of the apparent catalytic efficiency for LCAT activation, whereas deletion of the L e ~ ~ ~ - L e u l ~ ~ domain produced a 2-fold decrease. The differences in LCAT activation were due to differences in V,,, but not in Km *
DISCUSSION 94
67
43
Molecular Weight (kDa) FIGURE5 : Determination of the number of apo A-I molecules per rHDL particle by SDS-polyacrylamide gel scanning. rHDL particles, in which apo A-I molecules were cross-linked with bis(sulfosuccinimidyl) suberate, were subjected to electrophoresis on 10- 15% gradient SDS-polyacrylamide gels. The protein calibration mixture contained phosphorylase b (M,. 94 kDa), albumin ( M r 67 kDa), and ovalbumin ( M y 43 kDa). Because both plasma apo A-I and recombinant apo A-I produced rHDL particles that contained 2 apo A-I molecules per particle, only the gel scan for recombinant apo A-I is presented. residues Ksv = 0 and fa = 0. The Ksv values were 3 M-' for intact apo A-I (both plasma and recombinant apo A-I), apo A - I ( A G I U ' ~ ~ - L ~ Uand ~ ~ ~apo ) , A-I(AAla'90-Gln243) (Table 4) in rHDL as compared to 8 M-I for the apo A-I proteins free in solution, indicating that the accessibility of the Trp residues to the aqueous phase in the respective rHDL is reduced to the same extent. The Ksv value for apo A-I(ALeu44-Leu'26) in rHDL was 5 M-' and thus somewhat higher than for the other apo A-I proteins. However, apo A-I(ALeu44-Leu'26) contains only the Trp8 residue, and thus
In the present study, lipid binding of three deletion mutants of apo A-I, apo A - I ( A L ~ u ~ ~ - L ~ uapo ' ~ ~A-I(AGIu'~~), Leul'O), and apo A-I(AAla190-Gln243),was studied. Deletion of the Ala190-Gln243carboxy-terminal domain, but not of the L e ~ ~ ~ - L eamino-terminal u'~~ or the G l ~ ~ ~ ~ - L e u ' ' ~ central domains, resulted in a 10-fold slower interaction with phospholipids. However, the extent of in vitro phospholipid binding was similar for all apo A-I proteins, as demonstrated by comparable disc formation after mixing the apo A-I proteins and phospholipids at equal weight ratios. This was evidenced by a shift of the maximum Trp fluorescence to a shorter wavelength and by decreased accessibility of its Trp residues to I-. The sizes of rHDL constituted with apo A-I(AAla'90-Gln243), apo A - I ( A L ~ U ~ ~ - L ~ I and J ' ~ ~apo ), A - I ( A G I U ~ ~ ~ - Lwere ~ U larger ~ ~ ~ )than those with intact apo A-I. Furthermore, all mutant rHDL contained 4 apo A-I molecules per particle as compared to 2 for intact apo A-I. Circular dichroism scanning revealed 8 a-helices per intact apo A-I molecule, 5 per apo A - I ( A L ~ u ~ ~ - L ~6u per ' ~ ~apo ), A - I ( A G ~ U ' ~ ~ - L ~and U ' ~4~per ) , apo A-I(AAla'90-Gln243) molecule, as compared to the predicted numbers (Brasseur et al., 1990) of 8, 5, 6, and 6 a-helices, respectively. As the interaction of apolipoproteins with fluid interfaces is
Biochemistry, Vol. 34, No. 41, 1995 13341
Structure of Apo A-I in Mutant Reconstituted HDL Table 4: Fluorescence Properties of Apo A-I Proteins in rHDL Particles"
compound ~~~
properties wavelength of max fluorescence (nm) Stern-Volmer constant, KSV (M-I), for quenching of Trp fluorescence with Iquenchable fraction of Trp fluorescence (f,)
apo A-Ib 332 2.9 ?C 0.7
apo A-I(ALeu44-Leu126) 329 5.1 f 0.5'
0.61 f 0.08
0.62 f 0.05
apo A-Iapo A-I( A G l ~ l ~ ~ - L e u ' ~ ~(AAla'90-Gln243) ) 333 332 3.1 f 1.1 3.4 f 1.2 0.59 f 0.01
0.58 f 0.06
Data represent means i SD for 3 independent preparations. Values for plasma apo A-I and recombinant apo A-I were identical. Apo A-I(ALeu44-Leu'26) only contains the Trp* residue and not the Trp50, T ~ P ' ~and , Trp108residues. a
for optimal phospholipid and cholesterol presentation to the enzyme (Sparks et al., 1995). The reduced a-helical content of apo A-I(AAla19n-Gln243)in rHDL (as compared to the app Vmax app VmJapp K m predicted conformation I) and the 30% reduction of the (nmol of app K,,, [nmol of phospholipid surface that in the resulting rHDL particles is compound CE/h) (pM) CE/(h*pM)] covered with a-helices (Table 3) are in agreement with plasma apo A-I 6.0 12 0.50 alternative conformations of apo A-I(AAla190-Gln243) in recombinant apo A-I 6.2 14 0.44 apo A-I(ALeu"'-Leu'26) 2.7 11 0.20 rHDL. These differences are supporting the existence of a apo A - I ( A G I U ' ~ ~ - L ~ U ' ~0.44 ~) 11 0.04 hinged domain overlapping the segment apo A-I(AAla'90-Gln243) 0.59 13 0.05 (Figure 1, conformation 11) or overlapping the Ala124-His'62 a Data represent mean values for 2 independent measurements. V,, segment (Figure 1, conformation 111). The existence in apo and K, are derived from linear ( I = 0.99) Lineweaver-Burk plots of A-I of a hinged domain, constituted by a pair of central I/vo vs 1/[C] with [C] ranging from 10 to 40,uM. LCAT concentration a-helices, such as those located between residues Pro99 and was 50 nM. Leu143or Pro121and Pro165,has been proposed to explain driven by increased helix formation, the reduced a-helical the discontinuous sizes observed in HDL particles (Brouilcontent of apo A-I(AAla19n-Gln243)is indicative of an altered lette et al., 1984; Cheung et al., 1987) or the differences in conformation of this mutant in rHDL. immunoreactivity of monoclonal antibodies with lipid-free The LCAT activities of both apo A - I ( A G ~ L I ~ ~ ~ - Land ~ U ~ ' ~ )and lipid-bound forms of apo A-I (Calabresi et al., 1993; apo A-I(AAla19n-Gln243)were approximately 10-fold lower, Meng et al., 1993). Similar structures have also been whereas the activity of apo A - I ( A L ~ u ~ ~ - L ~was u I ~only ~) suggested by Breiter et al. (1991), on the basis of the X-ray 2-fold lower than that of intact apo A-I. Although LCAT determination of the structure of an insect apolipoprotein of activation may be sensitive to the size of the rHDL particles 18-20 kDa referred to as apolipoprotein I11 (Shapiro et al., (Barter et al., 1985; Dobiasova et al., 1991, 1992), this alone 1988). The fluorescence properties of apo A-I(AAla190cannot explain the differences in LCAT activity. Indeed, Gln243),showing that both the KSV and fa values are not whereas apo A-I(ALeu@-Leu126) rHDL particles are larger different from those of intact apo A-I, support more than intact apo A-I rHDL particle, their LCAT activities are conformation 111 than conformation 11. Recently an Nvery similar. Furthermore, whereas the sizes of the two other terminal proteolytic fragment of apo A-I was found to form mutant rHDL particles are similar to that of the first mutant rHDL with sizes and size distributions distinct from those rHDL particle, their LCAT activities are significantly lower. of intact apo A-I rHDL (Ji & Jonas, 1995) but with 6 Furthermore, differences in apo A-Uphospholipid ratios a-helices per apo A-I molecule in contact with POPC cannot explain the differences in LCAT activity. Therefore, phospholipids. The finding that these rHDL retained the the data suggest that deletion of the G l ~ ~ ~ ~ - L or e uthe "~ ability to activate LCAT further suggests that the carboxyAla19n-Gln243segments results in disruption of a critical terminal fragment of apo A-I is not necessary for the specific domain for LCAT activation. Domains in apo A-I that are activation of LCAT but that differences in the LCAT activity critical for LCAT activation may, by analogy with the of different rHDL produced with the same apo A-I proteins lipoprotein lipase/colipase system, contain regions which may rather be due to conformational changes of these apo mediate the binding of LCAT to the lipoprotein particle. A-I proteins in rHDL resulting from differences in composiAlternatively, they may contain regions that "activate" the tion. lipid substrates by promoting the correct orientation of the In conclusion, the present data suggest that any mutation sn-2 ester bond of phosphatidylcholine for presentation to in apo A-I that results in a disruption of the conformation the catalytic site of the enzyme and orientating the cholesterol of its central domain results in reduced LCAT activity. molecule within the surface monolayer in such a way that ACKNOWLEDGMENT its esterification is promoted. Finally, they may contain Dr. R. Brasseur is a senior scientist in the "Fonds de la regions that activate LCAT following a protein to protein Recherche Scientifique de Belgique". Dr. R. Vos is a interaction that results in a conformational change in the research associate of the Nationaal Fonds voor Wetenschapenzyme (Dolphin, 1992). In the present study, the differpelijk Onderzoek. We thank G. Vanderheeren (Interdisciences in LCAT activity appeared to be due to changes in plinary Research Center, University of Leuven at Kortrijk, the apparent V,,, values, which reflect the activated enzyme Belgium) for the CD measurements. concentration, and not to differences in K,, which are a function of the affinity of LCAT for rHDL (Bolin & Jonas, REFERENCES 1994). Thus, these data suggest that deletion of the G ~ u ~ ~ Anantharamaiah, ~ G. M., Jones, J. L., Brouillette, C. G., Schmidt, Leu17nor of the Ala190-Gln243domains may disrupt a critical C. F., Chung, B. H., Hughes, T. A,, Bhown, A. S., & Segrest, J. P. (1985) J. Bid. 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Table 5: Apparent Kinetic Parameters for the Reaction of LCAT with rHDL Particles"
~
~
~~
Holvoet et al.
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